The evolutionary molecular engineering is a science aiming at constructing a system that progressively evolves by repetition of three unit operations, “mutation”, “selection” and “amplification” utilizing the Darwin's evolution mechanism and applying the system in engineering. The evolutionary molecular engineering was theoretically proposed by Eigen et al. in 1984, and it is new biotechnology molecular design of functional biopolymers is performed by in vitro high-speed molecular evolution, that is, by investigating mechanisms of adaptive locomotion of biopolymers in a sequence space and optimizing them in laboratory experiments (Fushimi J. (1991) Kagaku, 61, 333-340; Fushimi J. (1992) Koza Shinka, vol. 6, University of Tokyo Press).
As one of important elemental techniques in the evolutionary molecular engineering, “assigning of genotype to phenotype” can be mentioned. The following three types of the “assigning of genotype to phenotype” are frequently adopted in the natural world or evolutionary molecular engineering (Fushimi J. (1999) Kagaku to Seibutsu, 37, 678-684):    (a) the ribozyme type in which a portion corresponding to a genotype and a portion corresponding to a phenotype are carried on the same molecule;    (b) the virus type in which a portion corresponding to a genotype and a portion corresponding to a phenotype form a complex; and    (c) the cell type in which a portion corresponding to a genotype and a portion corresponding to a phenotype are contained in a single compartment.The evolutionary molecular engineering of RNA is of (a) the ribozyme type, and (b) the virus type or (c) the cell type is contemplated for the evolutionary molecular engineering of proteins. In the 1990s, RNA evolutionary molecular engineering was developed by Joyce and Szostak et al. (Joyce, G. F. (1989) Gene, 82, 83; Szostak, J. W. & Ellington A. D. (1990) Nature, 346, 818), and in vitro experimental RNA systems (in vitro selection systems) utilizing (a) the ribozyme-type assigning technique were proposed. Subsequently, the in vitro virus method (Nemoto, N., Miyamoto-Sato, E., Yanagawa, H. (1997) FEBS Lett., 414, 405; Yanagawa, H., Nemoto, N., Miyamoto, E. (1998) WO98/16636), RNA-peptide fusion method (Roberts, R. W., Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA, 94, 12297), STABLE method (Doi, N. & Yanagawa, H. (1999) FEBS Lett., 457, 227) and so forth have been reported as in vitro experimental systems for proteins (in vitro selection systems) utilizing the virus-type technique of assigning a genotype to a phenotype in the protein evolutionary molecular engineering. In addition, various techniques of the virus-type evolutionary molecular engineering have been proposed so far including the phage display (Smith, G. P. (1985) Science, 228, 1315-1317; Scott, J. K. & Smith, G. P. (1990) Science, 249, 386-390), polysome display (Mattheakis, L. C. et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 9022-9026), library with encoding tags (Brenner, S. & Lerner, R. A. (1992) Proc. Natl. Acad. Sci. USA, 89, 5381-5383), Cellstat (Husimi, Y. et al. (1982) Rev. Sci. Instrum., 53, 517-522) and so forth.
The sequence space size, that is, size of a library, searchable in the evolutionary molecular engineering and post-genome functional analysis is important. As for the virus-type assigning molecules, the size of a library using is limited by a host cell when existing virus is utilized as in the case of phage display, since the virus parasites a cell. One the other hand, since the virus-type assigning molecules can be constructed in vitro in the aforementioned in vitro virus method (Nemoto, N., Miyamoto-Sato, E., Yanagawa, H. (1997) FEBS Lett., 414, 405; Yanagawa, H., Nemoto N. & Miyamoto E. (1998) WO98/16636), RNA-peptide fusion method (Roberts, R. W., Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA, 94, 12297) and so forth, these methods are theoretically expected as global searching methods for a sequence space comparable with the ribozyme-type technique. Further, in the evolutionary molecular engineering, not only the size of searchable sequence space, but also its diversity is important. The polysome display method (Mattheakis, L. C. & Dower, W. J. (1995) WO95/11922) is known, and this technique is suitable for a peptide with a short chain length, since nucleic acid and a protein are bonded by a noncovalent bond via ribosome in this technique. However, when the chain length becomes long like a protein, handling thereof becomes problematic, that is, diversity of library is limited due to the limited chain length of the genotype. As also for this problem, it is theoretically considered that no limitation is imposed on the chain length to be handled in the virus-type assigning molecules for the in vitro virus method, RNA-peptide fusion method and so forth. However, in order to actually construct a large-scale library and handle genotypes with a long chain length, several problems must be solved.
As described above, in principle, a large-scale library can be constructed by using virus-type assigning molecules in vitro as in the in vitro virus method, RNA-peptide fusion method and so forth. In practice, however, the size of the library depends on the efficiency of construction of the virus-type assigning molecules. The virus-type assigning molecules are constructed by bonding a spacer containing puromycin to a nucleic acid sequence containing protein information using a certain method to construct a genotype molecule and ligating it to a phenotype molecule (protein) on ribosome in a cell-free translation system. In this case, since a genotype molecule to which a spacer is not bonded, that is, a genotype molecule without puromycin, cannot be ligated to a phenotype molecule, the spacer binding efficiency determines the size of library. For example, in the RNA-peptide fusion method, a sprint and DNA ligase are used to ligate a DNA spacer. However, 1 random residue that does not exist in a template may be often added to the 3′-terminal end of the genotype molecule at the time of transcription. Thus, the sequence of the molecule does not match the sprint sequence, and hence ligation efficiency becomes poor. Accordingly, the sprint is modified, but much labor and cost are required. Further, in the in vitro virus method, RNA ligase is used to ligate a DNA spacer. Since RNA ligase does not require a sprint, it has no such a problem as that of the DNA ligase. However, it is known that RNA ligase originally has lower enzymatic activity compared with DNA ligase, and its ligation efficiency is also poor.
So far, in both of the in vitro virus method and the RNA-peptide fusion method, the usable cell-free translation system is limited to a rabbit reticulocyte cell-free translation system. Further, the virus-type assigning molecule construction efficiency in the rabbit reticulocyte cell-free translation system has remained as low as only 1% or lower (Roberts, R. W. & Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA, 94, 12297) or 10% or lower (Nemoto, N., Miyamoto-Sato, E., Yanagawa, H. (1997) FEBS Lett., 414, 405) of mRNA templates (genotype molecule) added to the cell-free translation system. Although the efficiency is increased to 20 to 40% by treatment after translation in the RNA-peptide fusion method (Liu, R., Barrick, E., Szoztak, J. W., Roberts, R. W. (2000) Methods in Enzymology, 318, 268-293), this requires much labor and time such as ordinary translation followed by addition of magnesium ions (Mg2+) and potassium ions (K+) and incubation at −20° C. for 2 days. In the rabbit reticulocyte cell-free translation system, in addition to the low assigning efficiency, mRNA stability is low, and therefore mRNA with a long chain cannot be handled. In contrast, in a wheat germ cell-free translation system, mRNA stability is favorable, and hence mRNA with a long chain can be handled. Therefore, it is desirable that a virus-type assigning molecule can be constructed on ribosome in the wheat germ cell-free translation system, but this has not been realized so far.
Among other factors determining the construction efficiency of virus-type assigning molecules, the most important one is the difference in translation efficiency of the genotype molecule. This can be expected to largely depend on sequences of a transcription promoter and a translation enhancer in the 5′ untranslated region (5′ UTR), 3′-end side sequence and so forth of the genotype molecule. However, there has been no report on examination of the relationship between the translation efficiency and the virus-type assigning molecule construction efficiency.